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Method for cloning and expressing target gene by homologous recombination

a technology of homologous recombination and target genes, which is applied in the field of cloning and expressing target genes by homologous recombination, can solve the problems of high cost and expensive enzymes, and achieve the effect of easy cloning of target genes and high speed

Inactive Publication Date: 2007-06-28
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] It is an object of the present invention to provide a method for cloning and expressing a target gene by homologous recombination, which can easily clone the target gene and express a protein at high speed, without needing complicated genetic engineering steps, such as the restriction enzyme digestion of a vector, and the ligation of an inserted fragment.

Problems solved by technology

However, in the prior genetic engineering technique, there are limitations in that inserted DNA and a vector should be cut with a restriction enzyme at the same recognition site, and the restriction enzyme should be selected from those which do not cut the inside of the insertion DNA or vector.
Also, the process of treating the inserted DNA and the vector with the restriction enzyme and ligase requires a skill, and much cost is incurred due to expensive enzymes.

Method used

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  • Method for cloning and expressing target gene by homologous recombination
  • Method for cloning and expressing target gene by homologous recombination
  • Method for cloning and expressing target gene by homologous recombination

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Transformed Cells Containing Expression Vectors pRMT, and pRMT & pKD46

[0084] To construct recombinant vector pRMT comprising a chloramphenicol-resistant gene (cat) having deletions of a ribosome recognition sequence and start codon required for the expression thereof as a first selection marker gene, and a kanamycin-resistant gene (kan) as a second selection marker gene, a homologous recombination technique was used, wherein a DNA fragment containing a PCR-amplified cat gene having deletion of a start codon was linked to a plasmid vector linearized with restriction enzymes (FIG. 1).

[0085] Specifically, in the homologous recombination technique, the linear vector and the linear DNA fragment containing sites homologous to each other, were introduced into recombinant E. coli (arabinose-induced pKD46 / E. coli JM109(DE3)) by electroporation to clon. This method has advantages over a conventional restriction enzyme-ligase technique, in that it has no restriction in selecti...

example 2

Processing of Target Gene

[0099] As a target gene for application to the recombinant vector pRMT constructed in Example 1, a red fluorescent protein gene (DsRed2) was used. The DsRed2 gene can be visually observed for the expression of protein by UV irradiation.

[0100] The red fluorescent protein gene (DsRed2) was amplified by PCR in the following manner. The red fluorescent protein gene (DsRed2) was amplified using a pDsRed2 (Clontech, USA) vector as a template with the following primers of SEQ ID NOS: 5 and 6.

SEQ ID NO: 5:5′-TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCCTCCTCCGAGAACG-3′SEQ ID NO: 6:5′-CTCATGTATATCTTATCTACAGGAACAGGTGGTGGCG-3′

[0101] Wherein, underlined regon represents forward homologous region and italic characters represent antisense sequence of start codon and forward ribosome binding site, respectively.

[0102] The primer of SEQ ID NO: 5 contains a base sequence homologous to a given length of the region downstream of the T7 promoter of pRMT, and the primer o...

example 3

Transformation Using Electroporation

[0107] The electro-competent cells containing pRMT & pKD46, prepared in Example 1, were transformed with 100 ng iTGR (a linear DNA fragment containing a DsRed2 gene) prepared in Example 2 by electroporation and then left to stand overnight at room temperature to induce homologous recombination. Because a chloramphenicol-resistant gene as the first selection marker gene is expressed in transformed cells where pRMT and iTGR were successfully homologously recombined with each other, the cells were spread on LB agar medium containing chloramphenicol so as to select colonies (FIG. 5).

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Abstract

A method for cloning and expressing a target gene by homologous recombination, and more particularly a method for cloning and expressing a target gene by homologous recombination, wherein a host cell transformed with a recombinant vector and a plasmid containing a recombinase system is introduced with a linear DNA fragment comprising a target gene and a sequence having homology to the recombinant vector. Because complicated genetic engineering steps, such as the restriction enzyme treatment and ligation of a vector and a target gene, are not required, the cloning of a gene can be performed without needing a high degree of skill, and enzyme cost can be reduced. The inventive method can be effectively used for the massive, high-speed cloning and protein expression of genes, and the disclosed pRMT-iTGR system can be used as an analytical means for improving high-efficiency recombinase.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority under 35 USC 119 of Korean Patent Application No. 10-2005-116672 filed Dec. 2, 2005. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to a method for cloning and expressing a target gene by homologous recombination, and more particularly to a method for cloning and expressing a target gene by homologous recombination, wherein a host cell transformed with a recombinant vector and a plasmid containing a recombinase system is introduced with a linear DNA fragment which contains a target gene and a sequence having homology to the recombinant vector. [0004] 2. Background of the Related Art [0005] DNA cloning refers to a technique for making a large amount of same gene family by linking a target gene to a self-replicable vector, such as plasmid, phage or cosmid, and introducing the vector into a host such as E. coli to proliferate the target gene. Cloning and sub...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N1/21C12N15/74
CPCC12N15/70C12N15/10C12N15/63C12N15/74C12N15/09
Inventor LEE, SEUNG-GOOSONG, JAE-JUNLEEHA, JAE-SEOK
Owner KOREA RES INST OF BIOSCI & BIOTECH
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